Heat-stable D-aminoacylase

ABSTRACT

The present invention provides a novel D-aminoacylase, as well as method for producing a D-amino acid using the same. In order to achieve the above objective, the present inventors have succeeded in purifying heat-stable D-aminoacylase from microorganisms belonging to the genus  Streptomyces  by combining various purification methods. Furthermore, the present inventors found that the purified heat-stable D-aminoacylase is useful in industrial production of D-amino acids. By utilizing the heat-stable D-aminoacylase, it is possible to readily and efficiently produce the corresponding D-amino acids from N-acetyl-DL-amino acids (for example, N-acetyl-DL-methionine, N-acetyl-DL-valine, N-acetyl-DL-tryptophan, N-acetyl-DL-phenylalanine, N-acetyl-DL-alanine, N-acetyl-DL-leucine, and so on).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/921,156, filed Aug. 2, 2001 now U.S. Pat. No. 6,596,528, which claimspriority under 35 U.S.C. 119 to Japanese Patent Application No.2002/234470, filed Aug. 2, 2000.

TECHNICAL FIELD

The present invention relates to a novel D-aminoacylase, as well asmethods for producing D-amino acids using the same.

BACKGROUND

Enzymes have excellent catalytic functions with substrate specificity,reaction specificity, and stereospecificity. Stereospecificity ofenzymes, with some exceptions, are nearly absolute.

Recent precise research has increased the importance of optically activesubstances for use in drugs, pesticides, feeds, and perfumes. Opticalisomers sometimes have quite different biological activities; forexample, D(R)-form thalidomide has no teratogenic activity, while itsL(S)-form shows strong teratogenicity. Thus, the practical use of thethalidomide racemate caused the drug injury incidents by thalidomide.Furthermore, if one enantiomer shows an effective biological activity,the other enantiomer sometimes not only has no activity but moreovercompetitively inhibits the activity of the effective enantiomer. As aresult, the biological activity of the racemate is reduced to half orless of the activity of the effective enantiomer. Accordingly, it isindustrially important to obtain (synthesize or optically resolve)optically pure enantiomers. For this objective, an effective procedurehas been used widely to optically resolve racemates synthesized. Inparticular, enzymatic optical resolution has drawn attention because itdoes not produce by-products and a bulk of liquid waste.

Generally, L-amino acids are widely and largely utilized in seasonings,food and feed additives, and infusions, and are thus very highlydemanded. L-amino acids have been produced mainly by direct fermentationusing microorganisms. Optical resolution in which N-acyl-DL-amino acidsare hydrolyzed with L-aminoacylases is also a known method for producingL-amino acids. It has been utilized to industrially produce L-aminoacids that are difficult to produce by fermentation. TheseL-aminoacylases are widely found in animals, plants, and microorganisms.They have been purified from various organisms, and their propertieshave been clarified. N-terminal amino acids of many proteins areconsidered to be N-acetylated in vivo. L-aminoacylases presumablyregenerate the N-acetyl-amino acids produced by decomposition ofproteins to amino acids. Among L-aminoacylases, an acylase that acts onN-acyl-L-glutamic acid is reported to be involved in argininebiosynthesis (Fruh, H., Leisinger, T.: J. Gen. Microb. 125, pp1(1981)).

In contrast, D-amino acids have not been a subject of interest for along time because they are nonprotein amino acids. D-amino acids wereknown to naturally occur only in small cyclic peptides, peptidoglycan ofbacterial cell walls, and peptide antibiotics. However, D-amino acidshave been demonstrated to be constituents of neuro-peptides and to existas binding forms in tooth enamel, the lens, and cerebral proteins,resulting in investigation of physiological significance and enzymaticsynthesis of D-amino acids.

At present, DL-amino acids have been optically resolved byphysicochemical, chemical, and enzymatic methods. The enzymatic methodsare the most convenient and industrially applicable for, for example,continuously producing L-methionine from N-acetyl-DL-methionine using abioreactor on which L-aminoacylase is immobilized. D-amino acids mayalso be produced using hydantoinase. The method involves a two-stepenzymatic reaction. The first reaction uses D-specific hydantoinase toconvert D,L-5-substituted-hydantoin, which is synthesized at low costfrom aldehyde analogues, to a D-carbamyl derivative. The second reactionuses D-amino acid carbamylase. Moreover, a method is known in whichD-aminoacylase hydrolyzes N-acetyl-DL-amino acids to produce D-aminoacids (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980),Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang, Y. J.: J.Enzyme Microb. Technol. 14, pp384 (1992)). Thus, D-aminoacylases areimportant for production of D-amino acids. However, their physiologicimportance and structural functions and so on remain to be resolved.

D-aminoacylase was first reported to be found in Pseudomonas sp. KT83isolated from soil by Kameda et al. in 1952 (Kameda, Y., Toyoura, H.,Kimura, Y. and Yasuda, Y.: Nature 170, pp888 (1952)). This enzymehydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, andD-alanine. Thereafter, D-aminoacylases derived from microorganisms werereported as follows:

Genus Pseudomonas (Kubo, K., Ishikura, T., and Fukagawa, Y.: J.Antibiot. 43, pp550 (1980); Kubo, K., Ishikura, T. and Fukagawa, Y.: J.Antibiot. 43, pp556 (1980); Kameda, Y., Hase, T., Kanatomo, S. and Kita,Y.: Chem. Pharm. Bull. 26, pp2698 (1978); Kubo, K., Ishikura, T. andFukagawa, Y.: J. Antibiot. 43, pp543 (1980));

Genus Streptomyces (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42,pp107 (1978); Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089(1980));

Genus Alcaligenes (Tsai, Y. C., Tseng, C. P., Hsiao, K. M. and Chen, L.Y.: Appl. Environ. Microbiol. 54, pp984 (1988); Yang, Y. B., Hsiao, K.M., Li, H., Yano, Y., Tsugita, A. and Tsai, Y. C.: Biosci. Biotech.Biochem. 56, pp1392 (1992); Yang, Y. B., Lin, C. S., Tseng, C. P., Wang,Y. J. and Tsai, Y. C.: Appl. Environ. Microbiol. 57, pp2767 (1991);Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang: Microb.Technol. 14, pp384 (1992); Moriguchi, M. and Ideta, K.: Appl. Environ.Microbiol. 54, pp2767 (1988); Sakai, K., Imamura, K., Sonoda, Y., Kido,H. and Moriguchi, M.: FEBS, 289, pp44 (1991); Sakai, K., Obata, T.,Ideta, K. and Moriguchi, M.: J. Ferment. Bioeng. 71, pp79 (1991); Sakai,K., Oshima, K. and Moriguchi, M.: Appl. Environ. Microbiol. 57, pp2540(1991); Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T. and Wakayama,M.: Biosci. Biotech. Biochem., 57, pp1145 (1993); Wakayama, M., Ashika,T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K. and Moriguchi,M.: J. Biochem. 118, pp204 (1995)); Moriguchi, M., Sakai, K., Miyamoto,Y. and Wakayama, M.: Biosci. Biotech. Biochem., 57, pp1149 (1993));

Genus Amycolatopsis (Japanese Patent Application No. Hei 9-206288);

Genus Sebekia (Japanese Patent Application No. Hei 10-089246); and

fungus (Japanese Patent Application No. Hei 10-228636).

Tsai et al. and Moriguchi et al. also clarified the characteristics ofD-aminoacylase derived from microorganisms belonging to the generaAlcaligenes and Pseudomonas and the amino acid and nucleotide sequencesof the enzymes. Moriguchi et al. found, by using different inducers,three types of D-aminoacylases in microorganisms belonging to the generaAlcaligenes and Pseudomonas (Wakayama, M., Katsumo, Y., Hayashi, S.,Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech. Biochem. 59,pp2115 (1995)).

Furthermore, Moriguchi et al. determined the nucleotide sequences ofthese D-aminoacylases derived from a microorganism belonging to thegenus Alcaligenes and compared them with L-aminoacylases derived fromBacillus stereothermophilus, human, and pig. The results demonstratedthat these D-aminoacylases have a low homology with L-aminoacylases(Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K. andMoriguchi, M.: Biosci. Biotech. Biochem., 59, pp2115 (1995)).

As to Actinomycetes, Sugie et al. reported D-aminoacylase of amicroorganism belonging to the genus Streptomyces (Sugie, M. and Suzuki,H.: Argric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H.:Argric. Biol. Chem. 44, pp1089 (1980)). However, the enzyme has not yetbeen purified, and its characteristics have not been well clarified.

The thermal stability of any of these known D-aminoacylases above arebelow 50° C., and the optimal temperature is below 50° C. NoD-aminoacylases with higher thermal stability is presently known. It iseconomically advantageous to use heat-stable D-aminoacylases, sincedurability of the enzyme rises with thermal stability. Moreover,application of heat-stable D-aminoacylases in the production of D-aminoacid possesses economic merit as well, since it is possible to set thereaction temperature high enough to elevate the concentration of thesubstrate and such due to the higher solubility.

SUMMARY

The object of the present invention is to isolate a heat-stableD-aminoacylase. Another object of the present invention is to providemethods for producing D-amino acids using the heat-stableD-aminoacylase.

In order to achieve the objectives above, the present inventors havesucceeded in purifying heat-stable D-aminoacylase from microorganismsbelonging to the genus Streptomyces by combining various purificationmethods. Furthermore, the present inventors found out that the purifiedheat-stable D-aminoacylase is useful in industrial production of D-aminoacids.

Thus, the present invention relates to the heat-stable D-aminoacylasebelow, as well as the use of the same. More specifically, the inventionprovides:

(1) A heat-stable D-aminoacylase having the following physicochemicalproperties of (a) to (c) below:

-   -   (a) action: the enzyme acts on N-acyl-D-amino acids to produce        the corresponding D-amino acid;    -   (b) thermal stability: the enzyme is stable at 55° C. when        heated at pH 7.5 for 60 minutes, but is inactivated at 70° C. or        more under the same condition;    -   (c) optimal temperature: under the condition of pH 7.5,        temperature around 60° C. is suited.

(2) The heat-stable D-aminoacylase of (1), which has also the followingphysicochemical properties of (d) to (g) below.

-   -   (d) molecular weight: a molecular weight of approximately 40,000        daltons measured by SDS-polyacrylamide gel electrophoresis;    -   (e) substrate specificity: the enzyme efficiently catalyzes the        reaction with N-acetyl-D-methionine, N-acetyl-D-tryptophan and        N-acetyl-D-phenylalanine, and catalyzes the reaction with        N-acetyl-D-valine, N-acetyl-D-alanine and N-acetyl-D-leucine,        but has substantially no catalytic activity for        N-acetyl-L-methionine, N-acetyl-L-valine, or        N-acetyl-L-phenylalanine;    -   (f) optimal pH: a pH about 7.0 is suited, when acted at 30° C.        for 60 minutes;    -   (g) effect of metal ion: the activity is accelerated with 1 mM        Co2+, but is markedly inhibited with 1 mM Cu2+.

(3) The heat-stable D-aminoacylase of (1) or (2), which is derived frommicroorganisms belonging to the genus Streptomyces.

(4) A heat-stable D-aminoacylase derived from Streptomycesthermonitrificans CS5-9, deposited under the accession No. FERM BP-7678.

(5) Streptomyces thermonitrificans CS5-9, deposited under the accessionNo. FERM BP-7678.

(6) A DNA encoding the heat-stable D-aminoacylase of any of (1) to (4).

(7) A method for producing the heat-stable D-aminoacylase of any of (1)to (4), said method comprising culturing a microorganism producing theheat-stable D-aminoacylase of any of (1) to (4) and recovering themicroorganism or the culture supernatant.

(8) A method for producing a D-amino acid, wherein said method comprisescontacting the heat-stable D-aminoacylase of any of (1) to (4) withN-acyl-DL-amino acid.

(9) A method for producing a D-amino acid, wherein said method comprisescontacting a microorganism producing the heat-stable D-aminoacylase ofany of (1) to (4), or a processed product thereof, with N-acyl-DL-aminoacid.

(10) The method of (8) or (9), wherein the N-acyl-DL-amino acid isN-acetyl-DL-methionine, N-acetyl-DL-valine, N-acetyl-DL-tryptophan,N-acetyl-DL-asparagine, N-acetyl-DL-phenylalanine, N-acetyl-DL-alanine,or N-acetyl-DL-leucine.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates purification by Butyl-Toyopearl column chromatographyof the D-aminoacylase of the present invention. The triangle denotes theabsorbance (at 280 nm); the line denotes the concentration of ammoniumsulfate (by % saturation). The active fraction is denoted by arrows(fraction numbers from 25 to 35). Additional parameters include:

gel volume: 180 ml;

resin: TK Butyl Toyopearl 650M;

equilibration and washing: 30% saturated ammonium sulfate containing 50mM phosphate buffer 800 ml; and

elution: 800 ml of buffer with a linear gradient of 30% to 0% saturatedammonium sulfate, each fraction contained 20 ml solution.

FIG. 2 illustrates purification by Butyl-Toyopearl column chromatography(2 nd) of the D-aminoacylase of the present invention. The triangledenotes the absorbance (at 280 nm); the line denotes the concentrationof ammonium sulfate (by % saturation). The active fraction is denoted byarrows (fraction numbers from 20 to 415). Additional parameters include:

gel volume: 50 ml

resin: TK Butyl Toyopearl 650M

equilibration and washing: 30% saturated ammonium sulfate containing 50mM phosphate buffer 800 ml

elution: 800 ml of buffer with a linear gradient of 30% to 0% saturatedammonium sulfate, each fraction contained 20 ml solution.

FIG. 3 illustrates purification by DEAE-Toyopearl column chromatographyof the D-aminoacylase of the present invention. The triangles denote theabsorbance (at 280 nm); the circles the activity (by mU/ml); and theline denotes the concentration of NaCl. Additional parameters include:

gel volume: 50 ml

equilibration and washing: 50 mM phosphate buffer 250 ml

elution: 250 ml of buffer with a linear gradient of 0 M to 0.5 M NaCl,each fraction contained 5 ml solution.

FIG. 4 illustrates purification of the D-aminoacylase of the presentinvention by gel filtration using HiPrep 16/60 Sephacryl S200 HR. Thetriangles denote the absorbance (at 280 nm), the circles the activity(by mU/ml). Additional parameters include:

gel volume: 320 ml

equilibration and washing: 640 ml of 50 mM phosphate buffer containing0.15 M NaCl

elution: the same buffer as above. Flow rate 0.5 ml/min

FIG. 5 illustrates the result of measurement of the molecular weight ofthe D-aminoacylase of the present invention by gel filtration. Theletters in the figure denote the following: A: gamma globulin (158,000);B: ovalbumin (44,000); C: myoglobin (17,000); D: vitamin B-12 (1,350).The circle denotes the position where the D-aminoacylase of the presentinvention was eluted and its molecular weight.

FIG. 6 illustrates the result of measurement of the molecular weight ofthe D-aminoacylase of the present invention by SDS-PAGE method.Phosphorylase B (97.4 K), serum albumin (66.1 K), ovalbumin (45 K), andcarbonic anhydrase (31 K) were used as the molecular weight marker. Theconcentration of separation gel was 10%. M: molecular weight marker; 1:crude enzyme solution; 2: Butyl-Toyopearl chromatography (first time);3: DEAE Toyopearl chromatography; 4: hiprep 10/60 Sephacryl S200 gelfiltration; 5: MonoQ HR 5/5.

FIG. 7 shows the thermal stability of the D-aminoacylase of the presentinvention. The standard reaction mixture solution without the substrate,N-acetyl-D-methionine, was preincubated for 30 minutes at eachtemperature, and was cooled thereafter to 0° C. immediately. Then,N-acetyl-D-methionine was added and the enzyme reaction was carried onfor 60 minutes. The remaining activity was calculated taking theactivity of the untreated reaction as 100%.

FIG. 8 shows the optimal reaction temperature of the D-aminoacylase ofthe present invention. The standard reaction mixture solution withoutthe enzyme was warmed to 30° C. before the enzyme reaction, and 15minutes of enzyme reaction was measured.

FIG. 9 shows the optimal reaction pH of the D-aminoacylase of thepresent invention. The triangles in the figure denote results withBis-Tris-HCl buffer (pH 5.0 to 7.0), and the circles with 50 mM Tris-HClbuffer (pH 7.5 to 10.0).

FIG. 10 shows the results of pH stability or the D-aminoacylase of thepresent invention. The enzyme was allowed to stand still for 20 minutesat each pH, at 4° C. Then, the activity was measured with standardreaction solution composition (pH 7.5) at 30° C. for 60 minutes. Theremaining activity was calculated, taking the activity of the reactionat pH 7.0 as 100%. 50 mM buffer of the following were used: opentriangle, citrate-NaOH (pH 3.0, 3.5); filled triangle, acetate-NaOH (pH4.0 to 5.0); open box, Bis-Tris-HCl (pH 5.0 to 7.0); filled box,Tris-HCl (pH 7.0 to 10.0); open circle, Borate-NaOH (pH 10.0 to 11.0).

DETAILED DESCRIPTION

The present invention relates to heat-stable D-aminoacylase. The“D-aminoacylase” of the present invention refers to enzymes thatcatalyze the production of organic acids and D-amino acids fromN-acyl-D-amino acid. The D-aminoacylase of the present invention has thephysicochemical properties of (a) to (c) below.

(a) action: the enzyme acts on N-acyl-D-amino acids to produce thecorresponding D-amino acid;

(b) thermal stability: the enzyme is stable at 55° C. when heated at pH7.5 for 60 minutes, but is inactivated at higher temperature than 70° C.under the same condition; and

(c) optimal temperature: under the condition of pH 7.5, temperaturearound 60° C. is suited.

The D-aminoacylase of the present invention preferably has also thephysicochemical properties of (d) to (g) below.

(d) molecular weight: a molecular weight of approximately 40,000 daltonsmeasured by SDS-polyacrylamide gel electrophoresis.

(e) substrate specificity: the enzyme efficiently catalyzes the reactionwith N-acetyl-D-methionine, N-acetyl-D-tryptophan andN-acetyl-D-phenylalanine, and catalyzes the reaction withN-acetyl-D-valine, N-acetyl-D-alanine and N-acetyl-D-leucine, but hassubstantially no catalytic activity for N-acetyl-L-methionine,N-acetyl-L-valine, or N-acetyl-L-phenylalanine.

(f) optimal pH: a pH about 7.0 is suited, when acted at 30° C. for 60minutes.

(g) effect of metal ion: the activity is accelerated with 1 mM Co²⁺, butis markedly inhibited with 1 mM Cu²⁺.

The activity of the D-aminoacylase of the present invention can betested by the following procedure. For example, an enzyme solution (100μl) is mixed and incubated with 50 mM Tris-HCl (pH 7.5) buffer (totalvolume: 500 μl) containing 20 mM substrate (various N-acetyl-D-aminoacids) at 30° C. for 60 minutes. By measuring the amounts of amino acidsynthesized by this reaction, enzymatic activities for respectivesubstrates can be compared with each other. Assay for amino acid isperformed by the TNBS (trinitrobenzenesulfonic acid) method or the HPLCmethod.

The enzyme activity is defined in units (U), assaying production ofD-methionine as the standard, wherein 1 U is defined as the amount ofenzyme that produces 1 μmol of D-methionine in 1 minutes at 30° C.Substrates other than D-methionine can be assayed by measuring theD-amino acid quantity produced in 1 minute at 30° C., as well. Thereactivity between enzymes or substrates can be compared to each otherutilizing this activity.

According to the results of the analysis discussed in detail below, theD-aminoacylase derived from the microorganisms belonging to the genusStreptomyces in the examples, were confirmed to catalyze especially wellreactions of the following substrates (see Table 2):

-   -   N-acetyl-D-methionine;    -   N-acetyl-D-tryptophan; and    -   N-acetyl-D-phenylalanine.

Also catalytic actions on the following substrates were confirmed:

-   -   N-acetyl-D-valine;    -   N-acetyl-D-alanine; and    -   N-acetyl-D-leucine.

On the other hand, substantially no catalytic activity was confirmed onthe following substrates:

-   -   N-acetyl-L-methionine;    -   N-acetyl-L-valine; and    -   N-acetyl-L-phenylalanine.

Thus, the present invention provides heat-stable D-aminoacylases thatcatalyze the reaction with N-acyl-D-amino acids to produce D-aminoacids. The heat-stable D-aminoacylase is stable when treated under a pHof 7.5 at 55° C. for 60 minutes, and is inactivated at a temperaturehigher than 70° C. under the same condition. The term “stable” as usedherein means that the activity is retained and includes comparativestability. Specifically, it can be referred to as being stable when atleast 20%, preferably more than 40%, more preferably more than 60% ofthe activity, as compared to the activity before the treatment, isretained. On the other hand, “inactivated” refers to a situation wherethe activity drops markedly or a situation where the activity is totallylost. Specifically, in the case where is the activity drops to less than10%, preferably less than 7%, and more preferably less than 5% of theactivity compared to the activity before the treatment, it can bedescribed as being inactivated. Activity can be measured as mentionedabove.

Moreover, the heat-stable D-aminoacylase of the present invention showsoptimal activity at a temperature around 60° C. when reacted at pH 7.5.The term “around 60° C.” herein means a temperature of 55 to 65° C.,preferably 57 to 63° C., and more preferably 58 to 62° C.

Further, the heat-stable D-aminoacylase of the present inventionpreferably shows a molecular weight of about 40,000 daltons measured bySDS-polyacrylamide gel electrophoresis. The term “about 40,000 daltons”encompasses a range of 35,000 to 45,000 daltons, preferably 37,000 to43,000 daltons, and more preferably 38,000 to 42,000 daltons. Moreover,the heat-stable D-aminoacylase of the present invention preferablyefficiently catalyzes the reaction with N-acetyl-D-methionine,N-acetyl-D-tryptophan and N-acetyl-D-phenylalanine, catalyzes thereaction with N-acetyl-D-valine, N-acetyl-D-alanine andN-acetyl-D-leucine, and has substantially no catalytic activity forN-acetyl-L-methionine, N-acetyl-L-valine and N-acetyl-L-phenylalanine.The term “efficiently catalyzes” as used herein means, substrates thatare catalyzed at average or above the average. And the term“substantially no catalytic activity” means that no detectable L-aminoacid is produced under the conditions above, or the activity calculatedas above is less than 10, preferably less than 5, and more preferablyless than 2 when taken the corresponding production activity of theD-amino acid to L-body is taken as 100.

Moreover, the heat-stable D-aminoacylase of the present invention showsoptimal activity at a pH of about 7.0 when activated at 30° C. for 60minutes. The term “a pH of about 7.0” encompasses a pH ranging from 6 to8, preferably 6.3 to 7.7, and more preferably 6.5 to 7.5. Further, theactivity is accelerated with 1 mM of Co²⁺, and is inhibited markedlywith 1 mM of Cu²⁺. The acceleration of the activity should be asignificant one. Furthermore, the term “marked inhibition of theactivity” refers to a situation wherein the activity is decreasedmarkedly or wherein the activity completely disappears. Specifically, itcan be mentioned, “the activity is inhibited markedly” when the activityis decreased to under 10%, preferably under 5%, and more preferablyunder 3% compared to the activity where no 1 mM Cu²⁺ exists. Theactivity can be measured and calculated as mentioned above.

There is no limitation on the derivation of the heat-stableD-aminoacylase of the present invention. Preferably the D-aminoacylaseof the present invention is derived from microorganisms. For example,microorganisms that can proliferate under a condition with highertemperature than 55° C. like thermophile, thermophilic bacteria, andsuch can be mentioned as microorganisms for the preparation of theheat-stable D-aminoacylase of the present invention. Bacteria of thegenus Bacillus (B. thermophilus, B. megaterium, B. coagulans, B.stearothermophilus, etc.), bacteria of the genus Clostridium (C.kluyveri and such), microorganisms of the genus Desulfotomaculum andsuch, as well as microorganisms of the genus Thermus (T. flavus, T.thermophilus, T. aguaticus, T. celer, etc.), methanogen(Methanobacterium, Methanococcus, Methanosarcina, etc.), lactic acidbacteria (Lactobacillus lactis, L. acidophilus, L. bulgaricus, L.delbrueckii, etc.), hydrogen bacteria, photosynthetic bacteria, and soon can be mentioned as thermophiles (thermophilic bacteria).Additionally, hyperthermophiles such as Pyrococcus furisus, Pyrococcussp., and Aeropyrum pernix and so on can be mentioned.

The heat-stable D-aminoacylase of the present invention is preferably aD-aminoacylase derived from a microorganism belonging to the genusStreptomyces. Streptomyces thermonitrificans is preferable asmicroorganisms belonging to the genus Streptomyces. For example,bacterial strain CS5-9 deposited as FERM BP-7678 can be mentioned assuch a microorganism.

The D-aminoacylase produced by the microorganism can be obtained byculturing the microorganism, and recovering the culture or the culturesupernatant. Either synthetic or natural media can be used, so long asthey contain proper amounts of a carbon source, nitrogen source,inorganic materials, and other nutrients. The culture media may beeither liquid or solid.

More specifically, examples of carbon sources include sugars such asglucose, fructose, maltose, galactose, starch, starch hydrolysate,molasses, and blackstrap molasses; natural carbohydrates such as wheat,barley, and corn; alcohols such as glycerol, methanol, and ethanol;fatty acids such as acetic acid, gluconic acid, pyruvic acid, and citricacid; hydrocarbons such as normal paraffin; and amino acids such asglycine, glutamine, and asparagine. One or more of the above carbonsources are used, depending on assimilability of the fungus used.Examples of nitrogen sources include organic nitrogen-containingcompounds such as meat extract, peptone, yeast extract, soybeanhydrolysate, milk casein, casamino acid, various amino acids, corn steepliquor, and other hydrolysates of animals, plants, and microorganisms;and inorganic nitrogen-containing compounds such as ammonia, ammoniumsalts such as ammonium nitrate, ammonium sulfate, ammonium chloride,nitrates such as sodium nitrate, and urea. One or more of the abovenitrogen sources are used, depending on assimilability of the fungus.

Furthermore, a minute amount of one or more inorganic salts can be used.Examples thereof include phosphates; hydrochlorides; nitrates; acetates;or similar salts of magnesium, manganese, potassium, calcium, sodium,copper, or zinc. Antifoaming agents, such as vegetable oil, surfactants,or silicon, may be added to the culture medium.

Culturing can be performed in the liquid medium containing theabove-described ingredients using the usual culture methods, such asshaking culturing, aerobic agitation culturing, continuous culturing, orfed-batch culturing.

Culturing conditions may be properly selected depending upon the fungalstrain and culture method, and are not particularly limited as long asthe fungi used can proliferate to produce D-aminoacylase. Ordinarily,the pH at the beginning of the cultivation is adjusted to pH 4 to 10,preferably to 6 to 8. A temperature that suits for the growth of themicroorganism is selected conveniently.

The culturing time is also not particularly limited so long as asufficient amount of fungal cells having the D-aminoacylase activity canbe obtained. The culturing is usually performed for 1 to 14 days,preferably for 1 to 3 days. The D-Aminoacylase produced and accumulatedwith gene expression can be recovered and isolated by the followingmethods.

When D-aminoacylase is intracellularly produced, the fungal cells arecollected by the method such as filtration or centrifugation after theculturing and washed with buffer, physiological saline, etc. The enzymecan then be extracted by disrupting the fungal cells using physicalmeans, such as freeze-thawing, ultrasonication, compression, osmotictreatment, or trituration; using biochemical means, such as cell walllysis with lysozyme; or using chemical means, such as surfactanttreatment. One or more of these treatments can be combined. The crudeD-aminoacylase thus obtained can be purified by a single or combinedfractionation means, including salting out; fractional precipitationwith organic solvents, etc.; various chromatographies, such assalting-out chromatography, ion-exchange chromatography, gel filtrationchromatography, hydrophobic chromatography, dye chromatography,hydroxylapatite chromatography, or affinity chromatography; andelectrophoresis, such as isoelectric focusing and nativeelectrophoresis. The above chromatographies can be performed using opencolumns or by means of medium-pressure or high-performance liquidchromatography (HPLC).

Specifically, the D-aminoacylase can be prepared for example by thepurification method described in the example below. That is, cultivationwith shaking is conducted in a medium for example, like 231 liquidculture medium (0.1% yeast extract, 0.1% meat extract, 1.0% maltose,0.2% N.Z.amine type A, pH 7.0), and harvested by centrifugation. Theobtained cell body is fragmented by ultrasonication with a sonicator,and the crude enzyme solution of the D-aminoacylase is obtained byrecovering the supernatant by centrifugation. Thereafter, precipitationtreatment using ammonium sulfate, desalting by gel filtration,Butyl-Toyopearl 650M hydrophobic chromatography, DEAE-Toyopearl 650Mion-exchange chromatography, Sephacryl S200 gel filtrationchromatography, MonoQ ion exchange chromatography is conducted to purifythe enzyme as a single band by SDS-polyacrylamide gel electrophoresis.

By utilizing the D-aminoacylase of the present invention, it is possibleto isolate the DNA encoding the same. The DNA encoding theD-aminoacylase of the present invention can be isolated, for example, bythe following method.

After purification of the enzyme of the present invention, theN-terminal amino acid sequence is analyzed. Then, it is digested withenzymes such as lysylendopeptidase and V8 protease, and the peptidefractions are purified by reverse phase liquid chromatography.Thereafter, many amino acid sequences can be determined by analyzingamino acid sequence by protein sequencer.

PCR primers are designed based on the determined amino acid sequence,and a part of the DNA encoding the D-aminoacylase of the presentinvention can be obtained by conducting PCR, using the genomic DNA orcDNA library of the enzyme-producing strain as the template, and a PCRprimer designed based on the amino acid sequence.

Moreover, DNA encoding the D-aminoacylase of the present invention canbe obtained by using the obtained DNA fragment as the probe, byinserting the restriction enzyme digest of the genomic DNA of theenzyme-producing strain into a phage or plasmid and such, transformingthe E. coli with it to obtain the library or cDNA library, andconducting colony hybridization, plaque hybridization, and so on.

It is also possible to obtain the DNA encoding the D-aminoacylase of thepresent invention by first analyzing the base sequence of the obtainedDNA fragment by PCR, and thereafter, designing a PCR primer to elongatethe known DNA outside. After digesting the genomic DNA of theenzyme-producing strain with an appropriate restriction enzyme, reversePCR is performed using the DNA as the template, by the self cyclizationreaction (Genetics 120, 621-623 (1988)), the RACE method (RapidAmplification of cDNA End, “PCR experimental manual” p25-33 HBJ press)and such. The DNA encoding a D-aminoacylase of the present inventioninclude not only the genomic DNA and cDNA cloned by the above-mentionedmethods but also chemically synthesized DNA.

The isolated DNA encoding the D-aminoacylase of the present invention isinserted into a known expression vector to provide aD-aminoacylase-expressing vector. Further, by culturing cellstransformed with the expression vector, the D-aminoacylase of thepresent invention can be obtained from the transformed cells.

There is no restriction on the microorganism to be transformed forD-aminoacylase expression in the present invention, so long as theorganism is capable of being transformed with the vector containing therecombinant DNA encoding the this D-aminoacylase and capable ofexpressing D-aminoacylase activity. Available microorganisms are thosefor which host-vector systems are available and include, for example:

bacteria such as the genus Escherichia, the genus Bacillus, the genusPseudomonas, the genus Serratia, the genus Brevibacterium, the genusCorynebacterium, the genus Streptococcus, and the genus Lactobacillus;

actinomycetes such as the genus Rhodococcus and the genus Streptomyces;

yeasts such as the genus Saccharomyces, the genus Kluyveromyces, thegenus Schizosaccharomyces, the genus Zygosaccharomyces, the genusYarrowia, the genus Trichosporon, the genus Rhodosporidium, the genusHansenula, the genus Pichia, and the genus Candida; and

fungi such as the genus Neurospora, the genus Aspergillus, the genusCephalosporium, and the genus Trichoderma; etc.

Procedure for the preparation of a transformant and construction of arecombinant vector suitable for a host can be carried out by employingtechniques that are commonly used in the fields of molecular biology,bioengineering, and genetic engineering (for example, see Sambrook etal., “Molecular Cloning”, Cold Spring Harbor Laboratories). In order toexpress the DNA encoding the D-aminoacylase of the present invention ina microorganism, it is necessary to introduce the DNA into a plasmidvector or phage vector that is stable in the microorganism and allow thegenetic information transcribed and translated. To do so, a promoter, aunit for regulating transcription and translation, is typically placedupstream of the 5′ end of the DNA encoding D-aminoacylase, andpreferably a terminator is placed downstream of the 3′ end of the DNA.The promoter and the terminator should be functional in themicroorganism to be utilized as a host. Available vectors, promoters,and terminators for the above-mentioned various microorganisms aredescribed in detail in “Fundamental Course in Microbiology (8): GeneticEngineering”, Kyoritsu Shuppan, specifically for yeasts, in “Adv.Biochem. Eng. 43, 75-102(1990)” and “Yeast 8, 423-488 (1992).”

For example, for the genus Escherichia, in particular, for Escherichiacoli, available plasmids include the pBR series and pUC series plasmids;available promoters include promoters derived from lac (derived fromβ-galactosidase gene), trp (derived from the tryptophan operon), tac andtrc (which are chimeras of lac and trp), P_(L) and P_(R) of λ phage,etc. Available terminators are derived from trpA, phages, rrnB ribosomalRNA, etc.

For the genus Bacillus, available vectors are the pUB110 series andpC194 series plasmids; the vectors can be integrated into hostchromosome. Available promoters and terminators are derived from apr(alkaline protease), npr (neutral protease), amy (α-amylase), etc.

For the genus Pseudomonas, there are host-vector systems developed forPseudomonas putida and Pseudomonas cepacia. A broad-host-range vector,pKT240, (containing RSF1010-derived genes required for autonomousreplication) based on TOL plasmid, which is involved in decomposition oftoluene compounds, is available; a promoter and a terminator derivedfrom the lipase gene (Unexamined Published Japanese Patent Application(JP-A) No. Hei 5-284973) are available.

For the genus Brevibacterium, in particular, for Brevibacteriumlactofermentum, available plasmid vectors include pAJ43 (Gene 39, 281(1985)). Promoters and terminators used for Escherichia coli can beutilized without any modification for Brevibacterium.

For the genus Corynebacterium, in particular, for Corynebacteriumglutamicum, plasmid vectors such as pCS11 (JP-A No. Sho 57-183799) andpCB101 (Mol. Gen. Genet. 196, 175(1984)) are available.

For the genus Streptococcus, plasmid vectors such as pHV1301 (FEMSMicrobiol. Lett. 26, 239 (1985)) and pGK1 (Appl. Environ. Microbiol. 50,94 (1985)) can be used.

For the genus Lactobacillus, plasmid vectors such as pAMβ1 (J.Bacteriol. 137, 614 (1979)), which was developed for the genusStreptococcus, can be utilized; and promoters that are used forEscherichia coli are also usable.

For the genus Rhodococcus, plasmid vectors isolated from Rhodococcusrhodochrous are available (J. Gen. Microbiol. 138, 1003 (1992)).

For the genus Streptomyces, plasmids can be constructed in accordancewith the method as described in “Genetic Manipulation of Streptomyces: ALaboratory Manual” (Cold Spring Harbor Laboratories (1985)) by Hopwoodet al. In particular, for Streptomyces lividans, pIJ486 (Mol. Gen.Genet. 203, 468-478, 1986), pKC1064 (Gene 103, 97-99 (1991)), andpUWL-KS (Gene 165, 149-150 (1995)) are usable. The same plasmids canalso be utilized for Streptomyces virginiae (Actinomycetol. 11, 46-53(1997)).

For the genus Saccharomyces, in particular, for Saccharomycescerevisiae, the YEp series, YEp series, YCp series, and YIp seriesplasmids are available; integration vectors (refer EP 537456, etc.),which are integrated into chromosome via homologous recombination withmulticopy-ribosomal genes, allow to introduce a gene of interest inmulticopy and the gene incorporated is stably maintained in themicroorganism; and thus, these types of vectors are highly useful.Available promoters and terminators are derived from genes encoding ADH(alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphatedehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK(phosphoglycerate kinase), ENO (enolase), etc.

For the genus Kluyveromyces, in particular, for Kluyveromyces lactis,available plasmids are those such as 2-μm plasmids derived fromSaccharomyces cerevisiae, pKD1 series plasmids (J. Bacteriol. 145,382-390(1981)), plasmids derived from pGK11 and involved in the killeractivity, KARS (Kluyveromyces autonomous replication sequence) plasmids,and plasmids (refer EP 537456, etc.) capable of being integrated intochromosome via homologous recombination with the ribosomal DNA.Promoters and terminators derived from ADH, PGK, and the like areavailable.

For the genus Schizosaccharomyces, it is possible to use plasmid vectorscomprising the ARS (autonomous replication sequence) derived fromSchizosaccharomyces pombe and auxotrophy-complementing selectablemarkers derived from Saccharomyces cerevisiae (Mol. Cell. Biol. 6, 80(1986)). Promoters such as ADH promoter derived from Schizosaccharomycespombe are usable (EMBO J. 6, 729 (1987)). In particular, pAUR224 iscommercially available from TaKaRa Shuzo Co., Ltd.

For the genus Zygosaccharomyces, plasmids originating from those such aspSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomycesrouxii are available; it is possible to use promoters such as the PHO5promoter derived from Saccharomyces cerevisiae and GAP-Zr(Glyceraldehyde-3-phosphate dehydrogenase) promoter (Agri. Biol. Chem.54, 2521 (1990)) derived from Zygosaccharomyces rouxii.

For the genus Hansenula, host-vector systems have been developed forHansenula polymorpha. Hansenula polymorpha-derived autonomousreplication sequences, HARS1 and HARS2, may be utilized as vectors, butthe replication sequences are relatively unstable, and accordinglymulticopy integration into chromosome is an effective way to ensurestable introduction of genes (Yeast 7, 431-443 (1991)). Promoters suchas that of the AOX (alcohol oxidase) gene, of which expression isinduced by methanol or the like, and a promoter from the FDH (formicacid dehydrogenase) are available.

For the genus Pichia, host-vector systems originating from autonomousreplication sequences (PARS1, PARS2) derived from Pichia have beendeveloped (Mol. Cell. Biol. 5, 3376 (1985)), and it is possible toemploy a highly efficient promoter, such as the methanol-inducible AOXpromoter, which is available for high-cell-density-culture (NucleicAcids Res. 15, 3859 (1987)).

For the genus Candida, host-vector systems have been developed forCandida maltosa, Candida albicans, Candida tropicalis, Candida utilis,etc. An autonomous replication sequence originating from Candida maltosahas been cloned (Agri. Biol. Chem. 51, 51, 1587 (1987)), and a vectorusing the sequence has been developed for Candida maltosa.

Further, a chromosome-integration vector with a highly efficientpromoter unit has been developed for Candida utilis (JP-A No. Hei08-173170).

For the genus Aspergillus, Aspergillus niger and Aspergillus oryzae haveintensively been studied among fungi, and thus plasmid vectors andchromosome-integration vectors are available, as well as promotersderived from an extracellular protease gene and amylase gene (Trends inBiotechnology 7, 283-287 (1989)).

For the genus Trichoderma, host-vector systems have been developed forTrichoderma reesei, and promoters such as that derived from anextracellular cellulase gene are available (Biotechnology 7, 596-603(1989)).

There are various host-vector systems developed for plants and animalsother than microorganisms; in particular, the systems include those ofinsect such as silkworm (Nature 315, 592-594 (1985)), and plants such asrapeseed, maize, potato, etc.

Various kinds of strains having D-aminoacylase production ability areencompassed by the present invention including mutant strains, mutation,and transformed strains with acquired production ability of theD-aminoacylase of the present invention made by the utilization of thegene manipulation technique.

A foreign gene contained in the transformant is induced underappropriate conditions given in a growth phase or after full growth. Forexample, in the case of lac promoter, addition of IPTG induces theexpression of the foreign gene that is connected downstream of thepromoter. As for a temperature-sensitive promoter, the culture isperformed at a temperature required for the expression.

Cultivation of the D-aminoacylase-producing strain (including organismstransformed by the D-aminoacylase expression vector), and purificationof the D-aminoacylase can be done by the same method as above. Theobtained D-aminoacylase can be utilized in the production of D-aminoacids. Moreover. The culture can be used in the D-amino acid productionas it is, or as a crude purification product by fracturing the cellbody. That is, the cell body cultured on the liquid medium or the platemedium is harvested, and the immobilized cell body, crude enzyme,immobilized enzyme and so on is prepared as occasion demands. TheD-amino acid production reaction system is constructed by contacting itwith the material, N-acyl-DL-amino acid. The N-acyl-DL-amino acid may beresolved in an appropriate solvent. The reaction can be carried out inan aqueous media with a buffering ability such as phosphate buffer, amixed media of aqueous media to which 1 to 100% water-soluble organicsolvent like methanol, ethanol, acetone and so on is mixed, and a twophase system of a aqueous media and non-water soluble solvent whichfails to dissolve in water, such as, n-hexane, ethyl acetate, isopropylacetate, n-butyl acetate, hexane, toluene, chloroform, and so on. In atwo-phase system, the D-aminoacylase, as well as the cell body and thereactant cell body is provided as it is, or as solutions in water orbuffers. Alternatively, the N-acyl-DL-amino acid can be provided to thereaction system resolved in an aqueous solvent such as water, buffer,ethanol, and so on. In this case, the D-aminoacylase, as well as thecell body or the reactant cell body constitutes a reaction system with asingular phase. Alternatively, the reaction of the present invention canbe carried out utilizing immobilized enzymes, membrane reactors andsuch. However, the configuration of the contact of the enzyme with thereaction solution is not limited to these specific examples. A reactionsolution is a convenient solvent, which provides a desirable environmentfor expression of the enzyme activity, to which the substrate isdissolved.

The heat-stable D-aminoacylase of the present invention has a propertyto produce D-amino acid, catalyzing reactions with variousN-acyl-D-amino acids under higher temperature regions as compared tothose of formerly known enzymes. Therefore, the solubility of thematerial and substrate elevates. And the concentration of thepreparation can be set higher. Therefore, using the heat-stableD-aminoacylase of the present invention it is possible to produce theD-amino acid in a much industrially efficient way. For example, D-aminoacid can be selectively produced by reacting D-aminoacylase of thisinvention with N-acyl-DL-amino acid, a mixture of D- and L-enantiomers.

N-acyl-DL-amino acids used in the present invention are not particularlylimited and can be selected from a wide variety of compounds. A typicalN-acyl-DL-amino acid can be represented by the formula (I):

where R₁ and R₂ may be identical or different and each represents ahydrogen atom or a substituted or unsubstituted hydrocarbon group;preferably, the hydrocarbon group represented by R₁ and R₂ is alkyl,alkenyl, alkynyl, cycloalkyl, aryl, or aralkyl, or the derivativethereof, and may be further substituted, provided that R₂ does notrepresent a hydrogen atom; and X is H, NH₄, or a metal ion.

The derivative used herein means those of alkyl, alkenyl, alkynyl,cycloalkyl, aryl, or aralkyl substituted with alkyl, alkenyl, alkynyl,cycloalkyl, aryl, aralkyl, hydroxy, halogen, amino, thio, methylthio, orthe like; or aryl or aralkyl of which aromatic ring moiety is aheterocycle comprising one or more nitrogen(s) or sulfur(s).

Specific examples of the hydrocarbon group contain from 1 to 10 carbonatoms, including linear or branched alkyl having 1 to 6 carbon atoms,such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl,t-butyl, n-pentyl, i-pentyl, n-hexyl, etc.; alkenyl having 1 to 6 carbonatoms such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl,2-pentenyl, 4-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, etc.; alkynylhaving 1 to 6 carbon atoms such as ethynyl, 1-propynyl, 2-pentynyl,etc.; aryl such as phenyl, naphthyl, etc.; cycloalkyl such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The substituentof the hydrocarbon group for R1 and R2 includes halogen; alkyl asdefined above; alkenyl as defined above; alkynyl as defined above; arylas defined above; heterocyclic such as piridyl, indole, quinolyl, etc.;amino; hydroxyl; thio; etc. The metal ion represented by X includessodium, patassium, etc.

Methyl, chloromethyl, phenyl, and aminomethyl, which may be substitutedwith the above substituent(s), can be mentioned as preferable R₂.Thiomethylethyl (for N-acyl-DL-methionine), isopropyl (forN-acyl-DL-valine), 3-indolylmethyl (for N-acyl-DL-tryptophan),carbamoylmethyl (for N-acyl-DL-asparagine), benzyl (forN-acyl-DL-phenylalanine), methyl (for N-acyl-DL-alanine), and2-methylpropyl (for N-acyl-DL-leucine) can be mentioned as preferableR₁.

N-acetyl-DL-amino acid is especially preferable as the substrate, towhich the heat-stable D-aminoacylase of the present invention is acted,and for example, N-acetyl-DL-methionine, N-acetyl-DL-valine,N-acetyl-DL-tryptophan, N-acetyl-DL-asparagine,N-acetyl-DL-phenylalanine, N-acetyl-DL-alanine, and N-acetyl-DL-leucinecan be preferably exemplified.

D-Aminoacylase used for producing D-amino acid in the present inventionincludes the partially purified enzyme as well as the purified one.Moreover, the present invention includes D-aminoacylase stabilized oninsoluble carrier by well-known method. Besides these enzyme proteins,the D-aminoacylase-producing microbe can also be used in the presentinvention. Namely, D-amino acid can be produced by directly reacting themicrobe capable of producing D-aminoacylase with N-acetyl-DL-amino acid.Further, it is possible to produce D-amino acids by reactingN-acetyl-DL-amino acids to the reactant microorganisms. The term“reactant microorganisms” includes those treated by physical treatment,such as freezing and thawing methods, ultrasonication, pressure, osmoticpressure difference, and grinding; biochemical treatment, such astreatment by cell wall lysing enzymes like lysozyme; or by chemicaltreatment, such as treatment by contacting to organic solvents likedetergents, toluene, xylene, acetone, and so on. Microorganisms withchanged permeability of the cell membrane by such treatment, orcell-free extracts in which cell bodies are disintegrated by treatmentwith glass beads or enzymes, or those partly purified are included in“reactant microorganisms”.

D-aminoacylase or microbes capable of producing the enzyme or itstreatment of present invention is reacted with N-acyl-D-amino acid underconditions suitable for the activity and stability of D-aminoacylase,and for the reactivity of the transformant. Some D-aminoacylases areactivated or inhibited by divalent metal ions, such as Zn²⁺, Ni²⁺, Co²⁺and Cu²⁺. When enzyme activity is inhibited by a divalent metal ion, achelating reagent such as EDTA can be added.

Though the concentration of the substrate, N-acetyl-DL-amino acid, isnot particularly limited, it is usually employed at a concentration ofabout 0.1 to 50 w/v %.

The substrate does not have to be completely dissolved in the reactionmedium. Additionally, the substrate can be added at the beginning of thereaction, but it is convenient to add it continuously or intermittentlyso that the substrate concentration in the reaction liquid does not risetoo high. The reaction may proceed earlier in most cases, where largequantities of D-aminoacylases are used; however, an amount of 1 U to1000 U/ml of the enzyme is normally used. The reaction temperature maybe set up according to the thermal stability of the enzyme, but isusually in the range of 5 to 70° C. Further, the reaction pH may be anyamount where the enzyme acts, but is usually a pH 4 to 10. The reactionmay be conducted with stirring or without stirring.

In general, an enzyme or a microorganism can be stabilized byimmobilization. Immobilization can be done by any known method, on asuitable carrier such as polyacrylamide gel, sulfur-containingpolysaccharide gel (carrageenan gel), alginic acid gel, chitin,cellulose or agar gel. Moreover, previous methods utilizecross-linking-treated carriers, such as glutaraldehyde. The timerequired for the reaction with the immobilized enzyme or microorganismdepends on the amounts of both D-aminoacylase and substrate. One skilledin the art can empirically optimize these conditions as the most idealones. Usually, ten- to one hundred-hour reaction efficiently produces adesired reaction product.

The D-amino acids produced can be recovered by a known method such asdirect crystallization by concentration or isoelectric precipitation,ion exchange resin treatment, membrane filtration, or the like. Forexample, D-tryptophan produced using N-acetyl-DL-tryptophan as asubstrate can be purified as follows. After the enzymatic reaction, thereaction mixture is contacted with a strongly acidic cation exchangeresin to adsorb D-tryptophan. The resin is washed with water andD-tryptophan is eluted with 0.5 N aqueous ammonia. After the eluate isconcentrated, the thus-obtained crude crystalline powder of D-tryptophanis dissolved in a small amount of 50% hot ethanol, decolorized withactivated charcoal, and cooled to obtain purified crystals ofD-tryptophan.

In the method of the present invention, D-valine can be purified asfollows. After the enzymatic reaction, the microbial cells are removedby centrifugation or the like, and the resulting supernatant is adjustedto pH 1 by adding 6 N hydrochloric acid. The precipitatedN-acetyl-L-valine is removed by centrifugation. The supernatant istreated with activated charcoal, adjusted to pH 7.0, then added to anH⁺-type strongly acidic cation exchanger (Amberlite IR-120B). Elution isperformed with 5% aqueous ammonia, and the resulting eluate is dried at80° C. under reduced pressure, thereby obtaining purified D-valine.

According to the present invention, a heat-stable D-aminoacylase derivedfrom Streptomyces thermonitrificans, as well as preparation method forD-amino acid using the same is provided. By utilizing the heat-stableD-aminoacylase, it is possible to produce readily and efficiently thecorresponding D-amino acids from N-acetyl-DL-amino acids (for example,N-acetyl-DL-methionine, N-acetyl-DL-valine, N-acetyl-DL-tryptophan,N-acetyl-DL-phenylalanine, N-acetyl-DL-alanine, N-acetyl-DL-leucine, andso on).

Any patents, patent applications, and publications cited herein areincorporated by reference.

The present invention is described in more detail with reference to thefollowing examples but is not to be construed to be limited thereto.

EXAMPLE 1

(1) Identification of the Strain

The properties of CS5-9, isolated from the soil of Shizuoka prefecture,was as follows. The color of the aerial hypha was gray and that of thesubstrate hypha had no color, and no diffusion melanin pigment was seen.The linkage form of the spore was spiral, and the sporophore wasgenerally straight but was rarely observed to be spiral in an ISP mediumcontaining glucose. It can utilize D-glucose, D-fructose, sucrose, andinositol, but it cannot grow on L-arabinose, D-xylose, D-mannitol,raffinose, or rhamnose. The LL-diaminopimelic acid is the main fattyacid composition of the cell wall. The identity analysis of the 16S rRNAbase sequence by direct sequencing with PCR revealed 100% identity withStreptomyces themonitrificans DSM 40579 (ISP 5579). Thus, the CS5-9strain was identified as belonging to the Streptomyces thermonitrificansbased on the ISP (International Streptomyces Project) and Bergey'smanual of Systematic Bacteriology (Volume 4) 1989.

This strain was deposited as “Streptomyces thermonitrificans CS5-9” withthe following depositary institution.

(i) Name and address of the depositary institution.

-   -   name: National Institute of Bioscience and Human-Technology,        Agency of Industrial Science and Technology, Ministry of        International Trade and Industry    -   address: (zip code 305-0046)        -   1-1-3, Higashi, Tsukuba-shi, Ibaraki 305, Japan

(ii) Date of deposit (first day when it was deposited)

-   -   Jul. 11, 2000

(iii) Accession No. FERM BP-7678

(2) Strain and Cultivation

The culture media to produce D-aminoacylase with Streptomycesthermonitrificans CS5-9 was prepared by pouring 120 ml of 231 liquidmedia (0.1% yeast extract (Oriental Yeast, Co., ltd.), 0.1% meatextract, 1.0% maltose, 0.2% N.Z.amine type A, pH 7.0) to each 500-mlvolume Sakaguchi flask, and sterilized in an autoclave (Speedclave,Kurihara medical instruments). The culture was conducted on a shaker at115 spm. for 42 hours at 50° C. 5 ml liquid media with the samecomposition was poured into a test tube and was sterilized in aSpeedclave to seed a volume of platinum loop from the slant (TBS agarmedia (0.4% polypeptone, 0.05% glucose, 0.5% NaCl, 0.25% K₂HPO₄, 2%agar, pH 7.3)) and to culture with shaking at 50° C. for 24 hours as apreculture.

After cultivation, centrifugation (Hitachi Koki, himac SCR20B, RPR10-2rotor) at 8,000 rpm (12,500×g) for 10 minutes at 4° C. was conducted toharvest the fungus. After washing the harvested fungus with 50 mMphosphate buffer (pH 7.0), it was centrifuged in the same rotor at 4,000rpm (3,130×g) for 10 minutes at 4° C. to obtain the fungus to be used.The obtained fungus was kept at −20° C.

(3) Method for D-aminoacylase Activity Measurement

The fungus obtained as above was disintegrated in 50 mM phosphate buffer(pH 7.0) by ultrasonication with a sonicator (Kubota, Insonator 201M) at190 W for 15 minutes. Then, it was subjected to centrifugation by acooling centrifuge (Hitachi Koki) using an RPM20-2 rotor at 17,500 rpm(39,000×g) for 15 minutes at 4° C. to obtain the supernatant. This wasused as the crude enzyme solution of D-aminoacylase.

The measurement of the enzyme activity was proceeded according to theTNBS method (Tokuyama, S., Hatano, K., and Takahashi, T.,Biosci.Biotech.Biochem. 58:24(1994)). That is, the sample containing theamino acid was added to 0.5 ml of solution (C) (0.1 M Na₂B₄O₇) to give atotal volume of 1.0 ml. 20 ml of 0.11 M TNBS solution was added withstirring immediately. The absorbance at 420 nm was measured after 5minutes.

D-methionine was measured colorimetrically using L-methionine as astandard, and the amount of enzyme-producing 1 μmol of D-methionine in 1minute at 30° C. was determined 1 unit.

The assay of the protein was conducted according to the method of Lowryusing BSA (Bovine Serum Albumin, Sigma) as the standard. That is, beforemeasurement, an alkaline copper solution, which is a 50:1 mixture ofsolution (A) (2% Na₂CO₃ (in 0.1 N NaOH)) and solution (B) (0.5% CuSO₄5H₂O (in 1% sodium citrate), was prepared and 1 ml of the alkalinecopper solution was added to the protein sample (5 to 50 μg ofproteins). Allowing it to stand still for 20 minutes at roomtemperature, 0.1 ml of phenol reagent (acid concentration 1 N) dilutedwith 2 volumes of distilled water was added and was left for 30 minutesat room temperature. Then, the absorbance at 750 nm was measured.

EXAMPLE 2

Purification of the heat-stable D-aminoacylase derived from Streptomycesthermonitrificans.

(1) Cultivation of the Fungus

The culture media for the production of D-aminoacylase by Streptomycesthermonitrificans CS5-9 strain was prepared by pouring 120 ml of 231liquid media (0.1% yeast extract (Oriental Yeast, Co., ltd.), 0.1% meatextract, 1.0% maltose, 0.2% N.Z.amine type A, pH 7.0) to each 500 mlvolume Sakaguchi flask, and sterilizing in a Speedclave (Kuriharamedical instruments). The culture was conducted on a shaker at 115 spm.for 42 hours at 50° C. 5 ml liquid media with the same composition waspoured into a test tube and was sterilized in a Speedclave to seed avolume of platinum loop from the slant (TBS agar media (0.4%polypeptone, 0.05% glucose, 0.5% NaCl, 0.25% K₂HPO₄, 2% agar, pH 7.3))and to culture with shaking at 50° C. for 24 hours as a preculture.

After cultivation, centrifugation (Hitachi Koki, himac SCR20B, RPR10-2rotor) at 8,000 rpm (12,500×g) for 10 minutes at 4° C. was conducted toharvest the fungus. After washing the harvested fungus with 50 mMphosphate buffer (pH 7.0), it was centrifuged in the same rotor at 4,000rpm (3,130×g) for 10 minutes at 4° C. to obtain the fungus to be used.The obtained fungus was kept at −20° C.

(2) Purification of D-aminoacylase

Purification procedure was carried out at 4° C. except otherwise stated.50 mM phosphate buffer (pH 7.0) was used as the buffer.

(2-1) Preparation of the Crude Enzyme Solution

980 g of wet fungus were suspended in 3 volumes of 50 mM phosphatebuffer (pH 7.0), and were ultrasonicated at 190 W for 25 minutes. Thenit was centrifuged (himac SCR20B HITACHI RPR10-2 rotor, 8,000 rpm(12,500×g), 15 min, 4° C.) to give a crude enzyme solution (2.35 L).

(2-2) Butyl-Toyopearl 650M Column Chromatography

To give a 30% saturated solution, ammonium sulfate was added to thecrude enzyme solution. The mixture was allowed to stand still for 1 hourat 0° C., then it was centrifuged (17,500 rpm ×30 minutes). Thesupernatant was absorbed on a Butyl-Toyopearl 650M column (200 ml),previously equilibrated with a buffer containing 30% saturated ammoniumsulfate. The column was washed with 1000 ml of the same buffer. Then waseluted with 1000 ml of the buffer decreasing the concentration ofammonium sulfate from 30% saturated to 0% to obtain the active fraction(100 ml). Ammonium sulfate was added to the active fraction to 60%saturation, and the mixture was allowed to stand still for 1 hour at 0°C., and was centrifuged (17,500 rpm ×30 minutes). After the precipitatewas rinsed with the buffer, it was diluted in the buffer (30 ml). Byconducting Butyl-Toyopearl 650M column chromatography, the enzyme of thepresent invention absorbed at a concentration of 30% saturated ammoniumsulfate, and no activity of the enzyme of the present invention wasobserved in the wash through fraction and washing fraction, but was inthe elution fraction (FIG. 1).

(2-3) Desalting Gel Filtration by Sephadex G-25

The above concentrated solution (30 ml) was gel filtrated to desalt withSephadexG-25 (100 ml), equilibrated in advance with 300 ml of buffercontaining 50 mM NaCl.

(2-4) Butyl-Toyopearl 650M Column Chromatography

To reach 30% saturation, ammonium sulfate was added to the desaltedactive fraction above (90 ml), absorbed on a Butyl-Toyopearl 650M column(50 ml), which was equilibrated in advance with the buffer containing30% ammonium sulfate, and washed with 250 ml of the same buffer. Elutionwith 250 ml of the buffer with a linear gradient from 30% saturated to0% ammonium sulfate was performed to obtain the active fraction (72 ml).Ammonium sulfate was added to 60% saturation to the active fraction, andit was allowed to stand still for one hour at 0° C., and thencentrifuged at 17,500 rpm for 30 minutes. After washing the precipitatewith the buffer, the fraction solubilized in 20 ml of the buffer.

(2-5) DEAE-Toyopearl Column Chromatography

The desalted fraction (35 ml) was absorbed on the DEAE-Toyopearl 650Mcolumn (50 ml), equilibrated in advance with a buffer, and washed with200 ml of the same buffer. Elution with 250 ml of the buffer with alinear gradient of 0 to 0.5 M NaCl, the enzyme of the present inventionwas considered being eluted at a concentration around 0.20 M NaCl, and5.0 ml of the active fraction was obtained. The result is shown in FIG.3.

(2-6) HiPrep 16/60 Sephacryl S200 HR Column Chromatography

The present chromatography was conducted at room temperature.

The active fraction obtained above (4.9 ml) was loaded on the HiPrep16/60 Sephacryl S200 HR, equilibrated in advance with a buffercontaining 0.15 M NaCl, eluted with the same buffer and divided intofractions of 1.0 ml. 3 ml of active fraction was obtained. By conductingHiPrep 16/60 Sephacryl S200 column chromatography (FPLC), the activefraction was obtained from the solution by eluting with 48 ml to 52 mlbuffer. The activity of the enzyme of the present invention reached thepeak at the elution volume of 50.5 ml. (FIG. 4)

The purification process is resumed in Table 1. The enzyme of thepresent invention was purified 107 folds, from 980 g of wet fungus byvarious chromatography at a yield of 0.16%. The specific activity was384 mU/mg.

TABLE 1 Purification of D-aminoacylase derived from S. thermonitrificansDegree of Purification Activity Total protein Yield purification step(mU) (mU/ml) (mU/mg) (mg) (%) (fold) Clude enzyme 24900 10.6 3.61 6900100 1 solution Butyl 4230 47.0 7.25 230 17 2 Toyopearl 650 M (1^(st))Butyl 2270 64.8 24.9 14.4 9.1 3.3 Toyopearl 650 M (2^(nd)) DEAE 591 12183.8 7.06 2.4 23 Toyopearl 650 M Hiprep 16/60 40.0 40.0 385 0.10 0.2 107SephacrylS200(3) SDS-Polyacrylamide Gel Electrophoresis

Separation gel solution 30% acrylamide mix.   6 ml H₂O  1.3 ml 0.75 MTris-HCl (pH 8.8)  7.5 ml 10% SDS  150 μl TEMED   12 μl

50 μl of 25% APS was added to the separation gel solution as preparedabove, and after stirring, it was poured into a gel plate until theupper surface of the gel solution reached the line 3 cm below the top ofthe plate, and H₂O was loaded onto the layer. After 10 to 20 minutes, asthe gel coagulated, the loaded H₂O was discarded.

Stacking gel solution 30% acrylamide mix. 0.75 ml H₂O  2.9 ml 0.75 MTris-HCl (pH 8.8) 3.75 ml 10% SDS   75 μl TEMED   6 μl

25 μl of 25% APS was added to the stacking gel solution as preparedabove, and after stirring, it was poured into the gel plate to the top,and a comb was inserted. The gel aggregated after about 20 to 60minutes. A volume of sample treating solution (0.125 M Tris-HCl (pH6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% BromophenolBlue) was added to the sample solution, diluted or concentrated asappropriate, treated in boiling water at 100° C. for 3 minutes. Andthen, applied 20 μl of it onto the gel. Tris-glycine buffer (25 mMTris-HCl (pH 8.4), 192 mM glycine, 0.1% SDS) was used as the buffer forelectrophoresis, and electrophoresis with 30 mA constant current wasconducted. The gel was stained with 0.25% Coomassie Brilliant Blue R-250solution for 1 hour, and was decolorized in a decolorizing solution(methanol:acetic acid:water(25:7.5:67.5) mixture).

By performing SDS-polyacrylamide gel electrophoresis with activefraction after various chromatographies, it was confirmed that theenzyme was purified to a single band with a molecular weight of 40,000after gel filtration.

The yield of the enzyme of the present invention after the purificationprocess was 0.2%, and was purified 107 times, showing a single band bySDS-PAGE.

EXAMPLE 3

Properties of heat-stable D-aminoacylase derived from Streptomycesthermonitrificans.

(1) Measurement of the Molecular Weight

The molecular weight was measured by (1) gel filtration method, and (2)SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method.

(1-1) Gel Filtration Method (FIG. 5)

“Superose 12 HR 10/30” (Pharmacia) was used as the column. “GelFiltration Standard” (Bio-Rad); thyroglobulin (670 K), gamma globulin(158 K), ovalbumin (44 K), myoglobin (17 K), vitamin B-12 (1.35 K) wasused as the marker of molecular weight. The flow rate was 0.25 ml/min.As a result, the enzyme of the present invention was determined to havea molecular weight of about 40,000 by gel electrophoresis.

(1-2) SDS-PAGE Method (FIG. 6)

Electrophoresis was conducted according to the method described beforeusing Mini-PROTEAN II electrophoresis apparatus (Bio-Rad) with aconstant current of 30 mA. Phosphorylase (97 K), serum albumin (66.1 K),ovalbumin (45 K), carbonic anhydrase (31 K) was used as the marker ofmolecular weight. After electrophoresis, the gel was stained withCoomassie Brilliant Blue, and compared with the molecular weight markedafter decolorizing with decolorizing solution I (100 ml acetic acid, 300ml methanol, 700 ml pure water) and decolorizing solution II (75 mlacetic acid, 50 ml methanol, 875 ml pure water). As a result, themolecular weight was estimated to be about 40,000.

Thus, the heat-stable D-aminoacylase derived from Streptomycesthermonitrificans was estimated to be a monomer with a molecular weightof about 40,000. The molecular weight of the enzyme of the presentinvention is smaller than that of the D-aminoacylase of genusAlcaligenes (MI-4 strain; 51,000, A-6 strain; 52,000, DA1 strain;55,000, DA181 strain; 58,000), and also was different from that of theD-aminoacylase of genus Streptomyces reported before (S. olivaceus;45,000).

(2) Substrate Specificity

The substrate specificity of the enzyme of the present invention wascompared taking the enzyme activity against N-acetyl-D-methionine as100%. N-acetyl-D-valine, N-acetyl-D-phenylalanine, N-acetyl-D-leucine,N-acetyl-D-tryptophan, N-acetvl-D-alanine N-acetyl-L-methionine,N-acetyl-L-leucine, and N-acetyl-L-valine was used as the substrate forcomparison. The enzyme activity was measured in a standard reactionsolution (total volume of 500 μl containing 100 μl of enzyme solution,and 400 μl of 50 mM Tris/HCl (pH 7.5) containing 20 mM of each substrateand 1 mM cobalt chloride at 30° C. for 60 minutes. The substratespecificity for N-acetyls of D-methionine, D-leucine, D-valine,D-tryptophan, D-alanine and D-phenylalanine is depicted in Table 2.

TABLE 2 Substrate specificity of D-aminoacylase Relative Activityactivity Substrate (20 mM) (mU/ml) (%) N-Ac-D-Met 40.6 100 N-Ac-D-Val14.7 40 N-Ac-D-Leu 8.93 21 N-Ac-D-Ala 16.9 40 N-Ac-D-Trp 48.4 105N-Ac-D-Phe 62.2 144 N-Ac-L-Met 0 0 N-Ac-L-Val 0 0 N-Ac-L-Phe 0 0

The enzyme of the present invention efficiently catalyzes reactions withN-acetyl-D-phenylalanine, N-acetyl-D-tryptophan andN-acetyl-D-methionine, and catalyzes also reactions withN-acetyl-D-leucine, N-acetyl-D-valine and N-acetyl-D-alanine. But didn'tcatalyze reactions with N-acetyl-L-methionine, N-acetyl-L-phenylalanineand N-acetyl-L-valine.

(3) Properties of the Enzyme

(3-1) Thermal Stability of the Enzyme

The enzyme solution was warmed to 30° C. to 70° C. for 30 minutes andwas cooled on ice immediately thereafter. Enzyme action of the treatedenzyme was measured in 50 mM Tris/HCl (pH 7.5) buffer (total volume of500 μl) at 30° C. for 60 minutes. The thermal stability of the enzyme ofthe present invention is shown in FIG. 7. The enzyme of the presentinvention was comparatively stable until 55° C., however the persistenceactivity decreased to 20% at 60° C., and was inactivated at 70° C.

(3-2) Optimal Reaction Temperature

Changing only the temperature to one of those selected from 30° C. to70° C., the enzyme reaction in 50 mM Tris/HCl (pH 7.5) buffer (totalvolume of 500 μl) for 15 minutes was performed. The optimal reactiontemperature of the enzyme of the present invention is shown in FIG. 8.The optimal temperature of the enzyme of the present invention wasestimated to be around 60° C.

(3-3) Optimal Reaction pH

Changing only the pH to one of those selected between pH 5.0 to pH 10.0,enzyme reaction at 30° C. for 60 minutes was performed. 50 mMBis-Tris-HCl buffer was used for the buffer with a pH between pH 5.0 to7.0, and 50 mM Tris-HCl buffer for that of pH 7.5 to 10.0. The optimalpH of the enzyme of the present invention is shown in FIG. 9. Theoptimal pH of the enzyme of the present invention was estimated to bearound pH 7.0.

(3-4) pH Stability

20 volumes of buffer with a pH between 3 to 11 were added to the enzymesolution. Enzyme reaction at pH 7.5, 30° C. for 60 minutes was performedafter incubating for 20 hours at 4° C. 50 mM citrate-NaOH buffer wasused for the buffer with pH 5.0 and 3.5, 50 mM Acetate-NaOH buffer forpH 4.0 to 5.0, 50 mM Bis-Tris-HCl buffer for pH 5.0 to 7.0, 50 mMTris-HCl buffer for pH 7.0 to 10.0, and 50 mM Borate-NaOH buffer for pH10.0 to 11.0. It was stable around a pH of 7.0. (FIG. 10)

(3-5) Effect of Various Metal Salt and Reagent

Various metal salt and various enzyme inhibitors were added to aconcentration of 1 mM to the enzyme reaction, and measurement of theenzyme reaction was performed. The enzyme reaction was conducted in 50mM Tris/HCl (pH 7.5) buffer (total volume of 500 μl). For the metal saltadded reaction, 1 mM of each metal salt or EDTA was added to thestandard reaction composition solution without the enzyme and waspreincubated at 30° C. for 5 minutes, then enzyme was added to react at30° C. for 60 minutes. For the enzyme inhibitor added reaction, thestandard reaction solution without the substrate was preincubated at 5minutes, then after adding 1 mM of each inhibitor substrate thesubstrate was added, and the enzyme reaction was performed at 30° C. for100 minutes. The relative activity was calculated taking activity ofenzyme actions without addition of inhibitors as 100%. The effect ofvarious metal salts and each inhibitor toward the enzyme of the presentinvention is set forth in Table 3 and Table 4.

TABLE 3 Effect of metal salt for D-aminoacylase Activity Relativeactivity Metal salt (mU/ml) (%) No addition 32.1 100 Al₂(SO₄)₂ 25.2 90BaCl₂ 30.6 95 CaCl₂ 31.1 97 CoCl₂ 54.4 168 CuCl₂ 1.0 3 FeCl₂ 20.4 63FeCl₃ 22.0 68 KCl 30.9 93 MgCl₂ 35.2 109 MnCl₂ 21.0 65 NaCl 33.1 103NiCl₂ 15.9 51 ZnCl₂ 22.2 68 SnCl₂ 32.8 102 EDTA 9.4 29

TABLE 4 Effect of inhibitors against D-aminoacylase Relative Activityactivity Reagent (mU/ml) (%) No addition 45.4 100 Hydroxylammoniumchloride 46.5 102 KI 11.3 24 Monoiodoacetic acid 37.9 84p-Chloromercuribenzoic acid 2.1 4 DTT 34.8 76 N-Ethylmaleimide 1.3 3 NaF45.2 99 2,2′-Bipyridyl 21.9 49 1,5-Diphenylcarbonylhydrazide 40.5 89Phenylmethylsulfonyl fluoride 29.5 70

The activity of the enzyme of the present invention was increased by theaddition of 1 mM Co²⁺, but was markedly inhibited by Cu²⁺. Additionally,the enzyme of the present invention was inhibited by SH reagent such asPCMB (p-chloromercuribenzoic acid) and N-ethylmaleimide. Furthermore, itwas also inhibited by metal chelating reagent, EDTA.

1. A method for producing a D-amino acid comprising contacting aheat-stable D-aminoacylase with an N-acyl-D-amino acid selected from thegroup consisting of N-acetyl-D-methionine, N-acetyl-D-tryptophan,N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-alanine, andN-acetyl-D-leucine to produce the corresponding D-amino acid, andrecovering said D-amino acid, wherein said D-aminoacylase has thefollowing physicochemical properties: (a) acts on N-acyl-D-amino acidsselected from the group consisting of N-acetyl-D-methionine,N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,N-acetyl-D-alanine, and N-acetyl-D-leucine to produce the correspondingD-amino acid; (b) thermal stability such that it is stable at 55° C.when heated at pH 7.5 for 60 minutes and is inactivated at 70° C. orhigher under the same condition; (c) optimal temperature of about 60° C.at pH 7.5; (d) molecular weight of about 40,000 daltons when determinedby SDS-polyacrylamide gel electrophoresis; (e) optimal pH of about 7.0at 30° C.; (f) metal ion effect such that the presence of 1 mM Co²⁺promotes activity; and (g) metal ion effect such that the presence of 1mM Cu²⁺ inhibits activity, wherein said D-aminoacylase is derived from amicroorganism belonging to the genus Streptomyces.
 2. A method forproducing a D-amino acid comprising contacting a microorganism producinga heat-stable D-aminoacylase with an N-acyl-D-amino acid selected fromthe group consisting of N-acetyl-D-methionine, N-acetyl-D-tryptophan,N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-alanine, andN-acetyl-D-leucine to produce the corresponding D-amino acid, andrecovering said D-amino acid, wherein said D-aminoacylase has thefollowing physicochemical properties: (a) acts on N-acyl-D-amino acidsselected from the group consisting of N-acetyl-D-methionine,N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,N-acetyl-D-alanine, and N-acetyl-D-leucine to produce the correspondingD-amino acid; (b) thermal stability such that it is stable at 55° C.when heated at pH 7.5 for 60 minutes and is inactivated at 70° C. ormere higher under the same condition; (c) optimal temperature of about60° C. at pH 7.5; (d) molecular weight of about 40,000 daltons whendetermined by SDS-polyacrylamide gel electrophoresis; (e) optimal pH ofabout 7.0 at 30° C.; (f) metal ion effect such that the presence of 1 mMCo²⁺ promotes activity; and (g) metal ion effect such that the presenceof 1 mM Cu²⁺ inhibits activity, wherein said D-aminoacylase is derivedfrom a microorganism belonging to the genus Streptomyces.
 3. The methodof claim 1, wherein said microorganism is Streptomyces thermonitrificansCS5-9, deposited under the accession number No. FERM BP-7678.
 4. Themethod of claim 1, wherein said D-aminoacylase is a purified enzyme. 5.The method of claim 1, wherein said D-aminoacylase is immobilized on asolid carrier.
 6. The method of claim 1, wherein said D-aminoacylase isin the form of a cell free extract of the microorganism.
 7. The methodof claim 1, wherein said D-aminoacylase is in the form of a cell lysate.8. The method of claim 2, wherein said microorganism is Streptomycesthermonitrificans CS5-9, deposited under the accession number No. FERMBP-7678.
 9. The method of claim 2, wherein said microorganism isimmobilized on a solid carrier.
 10. The method of claim 1, wherein theN-acyl-D-amino acid is present in a mixture with the correspondingN-acyl-L-amino acid.
 11. The method of claim 2, wherein theN-acyl-D-amino acid is present in a mixture with the correspondingN-acyl-L-amino acid.